Ayene I S, Koch C J, Krisch R E
Department of Radiation Oncology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Radiat Res. 1995 Oct;144(1):1-8.
A number of investigations have suggested that the widely observed oxygen enhancement of radiation-induced cell killing or intracellular DNA damage requires the presence of glutathione (GSH) or other thiols. We have adapted an in vitro model system to investigate the effects of GSH on radiation-induced DNA double-strand breaks (DSBs), lesions felt to be critical to cell death. Superhelical SV40 DNA, 25 micrograms/ml, was irradiated in air or nitrogen in the presence of 0-20 mM GSH and single-strand breaks (SSBs) and DSBs were measured using neutral gel electrophoresis/ethidium bromide fluorescence. Control experiments demonstrated that a substantial concentration of free SH was still present after irradiation. Dose-response curves for SSBs and DSBs in air or nitrogen were predominantly linear at all GSH concentrations tested from 0-20 mM, except for 20 mM GSH in nitrogen, indicating that both SSB and DSB formation are predominantly by one-hit mechanisms under these conditions. Dose-response curves for both SSBs and DSBs in nitrogen at 20 mM GSH closely tracked the corresponding linear curves in air for doses up to about 200 Gy, then reached a plateau at higher doses. Induction efficiencies in 20 mM GSH, calculated from these initial slopes for both SSBs and DSBs in nitrogen, were unexpectedly higher than the corresponding efficiencies in 5 mM GSH, suggesting additional damage, rather than the expected additional protection. The possible mechanism for a damaging effect from GSH is discussed. Oxygen enhancement ratios (OERs) were calculated from the slopes of dose-response curves. The OERs for SSBs did not differ substantially from those for DSBs at the same [GSH], contrary to the observations of Prise et al. (Radiat. Res. 134, 102-106, 1993). The OERs for SSBs and DSBs peaked at 6.5 and 8, respectively, at 5 mM GSH. These similarities suggest that the much lower OERs (2.5-3.0) generally reported for radiation killing of cells, which also typically contain about 5 mM GSH, cannot be accounted for by differences in OER between lethal lesions, represented by DSBs, and nonlethal lesions, represented by SSBs. In view of the present results, another possible explanation, that intracellular compounds other than reduced thiols are important in the chemical modification of the response of DNA to radiation, seems to be much more likely.
多项研究表明，辐射诱导的细胞杀伤或细胞内DNA损伤中广泛观察到的氧增强效应需要谷胱甘肽（GSH）或其他硫醇的存在。我们采用了一种体外模型系统来研究GSH对辐射诱导的DNA双链断裂（DSB）的影响，这种损伤被认为对细胞死亡至关重要。将25微克/毫升的超螺旋SV40 DNA在0-20 mM GSH存在的条件下于空气或氮气中进行辐照，并使用中性凝胶电泳/溴化乙锭荧光法测量单链断裂（SSB）和DSB。对照实验表明，辐照后仍存在大量游离SH。在0-20 mM测试的所有GSH浓度下，空气中或氮气中的SSB和DSB剂量反应曲线主要呈线性，但氮气中20 mM GSH除外，这表明在这些条件下，SSB和DSB的形成主要是通过单次打击机制。在20 mM GSH时，氮气中SSB和DSB的剂量反应曲线在剂量高达约200 Gy时紧密跟踪空气中相应的线性曲线，然后在更高剂量时达到平台期。根据氮气中SSB和DSB的这些初始斜率计算出的20 mM GSH中的诱导效率意外地高于5 mM GSH中的相应效率，这表明存在额外的损伤，而不是预期的额外保护。讨论了GSH产生损伤作用的可能机制。根据剂量反应曲线的斜率计算氧增强比（OER）。在相同的[GSH]下，SSB的OER与DSB的OER没有实质性差异，这与普里斯等人（《辐射研究》134, 102-106, 1993）的观察结果相反。在5 mM GSH时，SSB和DSB的OER分别在6.5和8时达到峰值。这些相似性表明，通常报道的细胞辐射杀伤的OER（2.5-3.0）低得多，而细胞通常也含有约5 mM GSH，这不能用DSB代表的致死性损伤和SSB代表的非致死性损伤之间的OER差异来解释。鉴于目前的结果，另一种可能的解释，即除了还原型硫醇之外的细胞内化合物在DNA对辐射反应的化学修饰中很重要，似乎更有可能。
